Disease Information:
Streptococcus galactiae causes infections in neonates and immunocompromised adults, especially elderly persons. This pathogen is one of the leading causes of invasive infections in non pregnant immunocompromised individuals and also causes bacteremia, septicaemia, meningitis, and pneumonia.
Colonization of the rectum and vagina of pregnant women with GBS which can cause inflammation and pain. A woman can have these bacteria in her body and not know it.
Infections from Group B Streptococcus (GBS) are not very serious in pregnant women and can readily be treated with antiobiotics. There is no way to prevent GS bacteria from being passed to a newborn at the time of birth. Some babes can still die as a result of complications from GBS.
Essentiality of this protein:
The essentially of this protein is very high due to the pyrophosphatase being a part of the serine/theonine protein kinase signaling cascade which phosphorylates serine/threonine residues and contributes to the virulence of S. agactiae. Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Complex of proteins?: no
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
none found
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): -- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/assay-library/ec-number-iii.html -- links to assay reagents (substrates) pages. http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/ionorgpyrophosph.pdf --- List cost and quantity of substrate reagents, supplier, and catalog #
$53 50mM Tris-HCl Buffer Supplier: Sigma-Aldrich #118553
$70 10mM Sodium pyrophosphate solution Supplier: bioPLUS #419201553
$20 10mM Magnesium chloride solution Supplier: Grainger Industries #7791186 $91 10% Ammonium molybdate solution Supplier: Fisher Scientific #7732185 $44 0.65 mM Phosphate standard Supplier: Sigma-Aldrich #7778770
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 2ENX
Current Inhibitors: no inhibitors Expression Information (has it been expressed in bacterial cells):
Induction times were optimized for both time and yield and both proteins were purified with the same protocol. Briefly, expression was performed using Luria Broth medium [10 g l−1 tryptone, 5 g l−1 yeast extract (Fluka Biochemica) and 5 g l−1 NaCl; J. T. Baker]. 50 µg ml−1 ampicillin (Sigma–Aldrich) was added to the culture medium. Escherichia coli BL21 (DE3) host cells (Novagen) were grown at 310 K to an OD600 of 0.5 and expression was then induced with 1 mM IPTG (isopropyl β-d-thiogalactopyranoside; Promega). Expression continued for 4 h at the lowered temperature of 303 K to minimize the formation of inclusion bodies. The cells were collected with centrifugation using a Beckman Coulter Avanti J-20 XP centrifuge and a JLA 8.1000 rotor (6240g at 277 K for 10 min).
The bacterial pellet from a 1 l growth was resuspended in 20 ml PBS (phosphate-buffered saline; 0.2 g l−1 KCl, 0.2 g l−1 KH2PO4, 8.0 g l−1 NaCl, 1.1 g l−1 Na2HPO4) buffer (reagents from J. T. Baker) and the solution was sonicated for 30 min using a Braun Labsonic sonicator at low power output with a 0.6 s duty cycle.
Purification Method:
After clarification by centrifugation using an Eppendorf 5810 R centrifuge and an F34-6-38 rotor (15 600g at 277 K for 20 min), the supernatant was applied onto a 10 ml Glutathione Sepharose 4B column (Amersham Biosciences), which was subsequently washed with several column volumes of PBS buffer. The inorganic pyrophosphatase was distinctly yellow, indicating bound metal ions, whereas the serine/threonine phosphatase was colourless. After binding, the protein on the column was treated overnight with thrombin protease (Amersham Biosciences) at room temperature using 100 units of protease per 10 mg of protein. The eluted protein concentrations were determined by Bradford assay (Bradford, 1976 ▶) and the purities and molecular weights of the enzymes were determined by SDS–PAGE (Laemmli, 1970 ▶). The gels were stained using Coomassie Brilliant Blue (Sigma Aldrich). The proteins were precipitated with ammonium sulfate (J. T. Baker) using 0.5 g ammonium sulfate per millilitre of protein solution and stored at 203 K. Image of protein (PyMol with features delineated and shown separately):
Fig 2. Protein 2ENX PyMOL image
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
Sequence A:
SKILVFGHQNPDSDAIGSSVAFAYLAKEAWGLDTEAVALGTPNEETAYVL
DYFGVQAPRVVESAKAEGVETVILTDHNEFQQSISDIKDVTVYGVVDHH
RVANFETANPLYMRLEPVGSASSIVYRMFKENGVSVPKELAGLLLSGLIS
DTLLLKSPTTHASDIPVAKELAELAGVNLEEYGLEMLKAGTNLSSKTAAEL
IDIDAKTFELNGEAVRVAQVNTVDINDILARQEEIEVAIQEAIVTEGYSDFVL
MITDIVNSNSEILALGSNMAKVEAAFEFTLENNHAFLAGAVSRKKQVVPQL
TESYNA
*length of your protein in Amino Acids: 642 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 69446.1 Molar Extinction coefficient of your protein at 280 nm wavelength: 37945 M-1 cm-1 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
results
Fig 3. Prediction of Transmembrane Regions and Orientation of 2ENX
*CDS Gene Sequence (paste as text only):
TTAAGCATTGTAGCTTTCTGTCAATTGAGGAACAACTTGTTTTTTGCGTGATACTGCCCCAGCAAGAAAAGCG
TGGTTATTTTCAAGTGTAAATTCAAAAGCTGCTTCAACTTTAGCCATATTTGAACCGAGAGCTAAAATCTCTGA
ATTTGAATTAACAATGTCAGTAATCATCAATACAAAATCAGAATAACCTTCTGTGACAATTGCTTCTTGAATAGC
AACTTCAATCTCTTCTTGACGTGCTAAAATATCGTTAATGTCCACAGTATTAACTTGTGCAACACGAACAGCTT
CTCCATTTAATTCAAATGTTTTAGCATCAATATCAATCAACTCCGCTGCAGTCTTACTTGAGAGGTTAGTACCTG
CTTTAAGCATTTCTAAACCATATTCTTCTAAGTTTACACCTGCAAGCTCTGCTAGTTCTTTTGCAACTGGAATAT
CTGAAGCATGAGTTGTTGGTGATTTCAAAAGAAGAGTATCTGAAATCAAACCTGATAATAATAAACCAGCCAA
CTCTTTGGGTACTGATACTCCATTTTCCTTAAACATACGGTAAACAATCGATGATGCTGAGCCAACTGGTTCCA
AACGCATATATAACGGATTGGCAGTCTCAAAGTTAGCAACACGATGATGATCAACAACACCATAAACTGTCACA
TCTTTAATATCAGAAATAGATTGTTGGAATTCGTTATGGTCAGTTAAAATGACTGTCTCTACACCCTCTGCTTTT
GCAGATTCTACTACTCGAGGTGCTTGAACACCAAAATAATCTAAAACATATGCTGTTTCTTCATTTGGAGTTCC
TAAAGCAACGGCCTCTGTGTCAAGCCCCCAAGCTTCTTTAGCTAGATATGCAAAAGCAACTGATGATCCAATA
GCATCTGAATCTGGATTTTGGTGACCAAAGACTAAAATTTTAGACAT
*GC% Content for gene: 37% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Manganese-dependent inorganic pyrophosphatase/ppaC SAK_1430
NCBI Gene # or RefSeq#: 827159899
Protein ID (NP or XP #) or Wolbachia#: KLL66391.1
Organism (including strain): Streptococcus galactiae
Etiologic Risk Group (see link below):/ 2
Disease Information:
Streptococcus galactiae causes infections in neonates and immunocompromised adults, especially elderly persons. This pathogen is one of the leading causes of invasive infections in non pregnant immunocompromised individuals and also causes bacteremia, septicaemia, meningitis, and pneumonia.
Colonization of the rectum and vagina of pregnant women with GBS which can cause inflammation and pain. A woman can have these bacteria in her body and not know it.
Infections from Group B Streptococcus (GBS) are not very serious in pregnant women and can readily be treated with antiobiotics. There is no way to prevent GS bacteria from being passed to a newborn at the time of birth. Some babes can still die as a result of complications from GBS.
Link to TDR Targets page (if present): none found
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
http://www.ncbi.nlm.nih.gov/protein/KLL63891.1
Essentiality of this protein:
The essentially of this protein is very high due to the pyrophosphatase being a part of the serine/theonine protein kinase signaling cascade which phosphorylates serine/threonine residues and contributes to the virulence of S. agactiae.
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Complex of proteins?: no
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
none found
*EC#: 3.6.1.1
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=3.6.1.1
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/assay-library/ec-number-iii.html
-- links to assay reagents (substrates) pages.
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/ionorgpyrophosph.pdf
--- List cost and quantity of substrate reagents, supplier, and catalog #
$53 50mM Tris-HCl Buffer Supplier: Sigma-Aldrich #118553
$70 10mM Sodium pyrophosphate solution Supplier: bioPLUS #419201553
$20 10mM Magnesium chloride solution Supplier: Grainger Industries #7791186
$91 10% Ammonium molybdate solution Supplier: Fisher Scientific #7732185
$44 0.65 mM Phosphate standard Supplier: Sigma-Aldrich #7778770
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2ENX
Current Inhibitors: no inhibitors
Expression Information (has it been expressed in bacterial cells):
Induction times were optimized for both time and yield and both proteins were purified with the same protocol. Briefly, expression was performed using Luria Broth medium [10 g l−1 tryptone, 5 g l−1 yeast extract (Fluka Biochemica) and 5 g l−1 NaCl; J. T. Baker]. 50 µg ml−1 ampicillin (Sigma–Aldrich) was added to the culture medium. Escherichia coli BL21 (DE3) host cells (Novagen) were grown at 310 K to an OD600 of 0.5 and expression was then induced with 1 mM IPTG (isopropyl β-d-thiogalactopyranoside; Promega). Expression continued for 4 h at the lowered temperature of 303 K to minimize the formation of inclusion bodies. The cells were collected with centrifugation using a Beckman Coulter Avanti J-20 XP centrifuge and a JLA 8.1000 rotor (6240g at 277 K for 10 min).
The bacterial pellet from a 1 l growth was resuspended in 20 ml PBS (phosphate-buffered saline; 0.2 g l−1 KCl, 0.2 g l−1 KH2PO4, 8.0 g l−1 NaCl, 1.1 g l−1 Na2HPO4) buffer (reagents from J. T. Baker) and the solution was sonicated for 30 min using a Braun Labsonic sonicator at low power output with a 0.6 s duty cycle.
Purification Method:
After clarification by centrifugation using an Eppendorf 5810 R centrifuge and an F34-6-38 rotor (15 600g at 277 K for 20 min), the supernatant was applied onto a 10 ml Glutathione Sepharose 4B column (Amersham Biosciences), which was subsequently washed with several column volumes of PBS buffer. The inorganic pyrophosphatase was distinctly yellow, indicating bound metal ions, whereas the serine/threonine phosphatase was colourless. After binding, the protein on the column was treated overnight with thrombin protease (Amersham Biosciences) at room temperature using 100 units of protease per 10 mg of protein. The eluted protein concentrations were determined by Bradford assay (Bradford, 1976 ▶) and the purities and molecular weights of the enzymes were determined by SDS–PAGE (Laemmli, 1970 ▶). The gels were stained using Coomassie Brilliant Blue (Sigma Aldrich). The proteins were precipitated with ammonium sulfate (J. T. Baker) using 0.5 g ammonium sulfate per millilitre of protein solution and stored at 203 K.
Image of protein (PyMol with features delineated and shown separately):
Fig 2. Protein 2ENX PyMOL image
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
Sequence A:
SKILVFGHQNPDSDAIGSSVAFAYLAKEAWGLDTEAVALGTPNEETAYVL
DYFGVQAPRVVESAKAEGVETVILTDHNEFQQSISDIKDVTVYGVVDHH
RVANFETANPLYMRLEPVGSASSIVYRMFKENGVSVPKELAGLLLSGLIS
DTLLLKSPTTHASDIPVAKELAELAGVNLEEYGLEMLKAGTNLSSKTAAEL
IDIDAKTFELNGEAVRVAQVNTVDINDILARQEEIEVAIQEAIVTEGYSDFVL
MITDIVNSNSEILALGSNMAKVEAAFEFTLENNHAFLAGAVSRKKQVVPQL
TESYNA
Sequence B:
SKILVFGHQNPDSDAIGSSVAFAYLAKEAWGLDTEAVALGTPNEETAYVL
DYFGVQAPRVVESAKAEGVETVILTDHNEFQQSISDIKDVTVYGVVDHH
RVANFETANPLYMRLEPVGSASSIVYRMFKENGVSVPKELAGLLLSGLIS
DTLLLKSPTTHASDIPVAKELAELAGVNLEEYGLEMLKAGTNLSSKTAAE
LIDIDAKTFELNGEAVRVAQVNTVDINDILARQEEIEVAIQEAIVTEGYSDF
VLMITDIVNSNSEILALGSNMAKVEAAFEFTLENNHAFLAGAVSRKKQVV
PQLTESYNA
*length of your protein in Amino Acids: 642
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 69446.1
Molar Extinction coefficient of your protein at 280 nm wavelength: 37945 M-1 cm-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Fig 3. Prediction of Transmembrane Regions and Orientation of 2ENX
*CDS Gene Sequence (paste as text only):
TTAAGCATTGTAGCTTTCTGTCAATTGAGGAACAACTTGTTTTTTGCGTGATACTGCCCCAGCAAGAAAAGCG
TGGTTATTTTCAAGTGTAAATTCAAAAGCTGCTTCAACTTTAGCCATATTTGAACCGAGAGCTAAAATCTCTGA
ATTTGAATTAACAATGTCAGTAATCATCAATACAAAATCAGAATAACCTTCTGTGACAATTGCTTCTTGAATAGC
AACTTCAATCTCTTCTTGACGTGCTAAAATATCGTTAATGTCCACAGTATTAACTTGTGCAACACGAACAGCTT
CTCCATTTAATTCAAATGTTTTAGCATCAATATCAATCAACTCCGCTGCAGTCTTACTTGAGAGGTTAGTACCTG
CTTTAAGCATTTCTAAACCATATTCTTCTAAGTTTACACCTGCAAGCTCTGCTAGTTCTTTTGCAACTGGAATAT
CTGAAGCATGAGTTGTTGGTGATTTCAAAAGAAGAGTATCTGAAATCAAACCTGATAATAATAAACCAGCCAA
CTCTTTGGGTACTGATACTCCATTTTCCTTAAACATACGGTAAACAATCGATGATGCTGAGCCAACTGGTTCCA
AACGCATATATAACGGATTGGCAGTCTCAAAGTTAGCAACACGATGATGATCAACAACACCATAAACTGTCACA
TCTTTAATATCAGAAATAGATTGTTGGAATTCGTTATGGTCAGTTAAAATGACTGTCTCTACACCCTCTGCTTTT
GCAGATTCTACTACTCGAGGTGCTTGAACACCAAAATAATCTAAAACATATGCTGTTTCTTCATTTGGAGTTCC
TAAAGCAACGGCCTCTGTGTCAAGCCCCCAAGCTTCTTTAGCTAGATATGCAAAAGCAACTGATGATCCAATA
GCATCTGAATCTGGATTTTGGTGACCAAAGACTAAAATTTTAGACAT
*GC% Content for gene: 37%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):